Photodynamic Therapy of Tumors Can Lead to
Development of Systemic Antigen-Specific Immune
Response
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Mroz P, Szokalska A, Wu MX, Hamblin MR (2010)
Photodynamic Therapy of Tumors Can Lead to Development of
Systemic Antigen-Specific Immune Response. PLoS ONE 5(12):
e15194. doi:10.1371/journal.pone.0015194
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http://dx.doi.org/10.1371/journal.pone.0015194
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Photodynamic Therapy of Tumors Can Lead to
Development of Systemic Antigen-Specific Immune
Response
Pawel Mroz1,2, Angelika Szokalska1,3, Mei X. Wu1,2,4, Michael R. Hamblin1,2,4*
1 Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 2 Department of Dermatology, Harvard Medical
School, Boston, Massachusetts, United States of America, 3 Department of Immunology, Medical University of Warsaw, Warsaw, Poland, 4 Harvard-MIT Division of Health
Sciences and Technology, Cambridge, Massachusetts, United States of America
Abstract
Background: The mechanism by which the immune system can effectively recognize and destroy tumors is dependent on
recognition of tumor antigens. The molecular identity of a number of these antigens has recently been identified and
several immunotherapies have explored them as targets. Photodynamic therapy (PDT) is an anti-cancer modality that uses a
non-toxic photosensitizer and visible light to produce cytotoxic reactive oxygen species that destroy tumors. PDT has been
shown to lead to local destruction of tumors as well as to induction of anti-tumor immune response.
Methodology/Principal Findings: We used a pair of equally lethal BALB/c colon adenocarcinomas, CT26 wild-type
(CT26WT) and CT26.CL25 that expressed a tumor antigen, b-galactosidase (b-gal), and we treated them with vascular PDT.
All mice bearing antigen-positive, but not antigen-negative tumors were cured and resistant to rechallenge. T lymphocytes
isolated from cured mice were able to specifically lyse antigen positive cells and recognize the epitope derived from betagalactosidase antigen. PDT was capable of destroying distant, untreated, established, antigen-expressing tumors in 70% of
the mice. The remaining 30% escaped destruction due to loss of expression of tumor antigen. The PDT anti-tumor effects
were completely abrogated in the absence of the adaptive immune response.
Conclusion: Understanding the role of antigen-expression in PDT immune response may allow application of PDT in
metastatic as well as localized disease. To the best of our knowledge, this is the first time that PDT has been shown to lead
to systemic, antigen- specific anti-tumor immunity.
Citation: Mroz P, Szokalska A, Wu MX, Hamblin MR (2010) Photodynamic Therapy of Tumors Can Lead to Development of Systemic Antigen-Specific Immune
Response. PLoS ONE 5(12): e15194. doi:10.1371/journal.pone.0015194
Editor: R. Lee Mosley, University of Nebraska Medical Center, United States of America
Received August 6, 2010; Accepted October 29, 2010; Published December 10, 2010
Copyright: ß 2010 Mroz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Supported by US NIH National Cancer Institute (grants RO1CA/AI838801 and R01AI050875 to MRH). P.M. was partly supported by Genzyme-Partners
Translational Research Grant. A.S. was supported by the Foundation for Polish Science, International Union Against Cancer and European Structural Fund:
"Mazowieckie Stypendium Doktoranckie." The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests: Pawel Mroz was partly supported by a Genzyme-Partners Translational Research Grant. This a peer-reviewed competitive grant open to
Partners investigators and funded by Genzyme. The project that was funded was nothing to do with the present paper (it was to do with kidney cancer and antiTGF-beta antibody) but it did support Mroz salary. We confirm that this did not alter our adherence to all the PLoS ONE policies on sharing data and materials, as
detailed online in our guide for authors.
* E-mail: hamblin@helix.mgh.harvard.edu
from viruses [12,13,14,15,16]. The immunotherapeutic strategies that target tumor antigens have been successfully developed
and tested in preclinical studies and early-phase clinical trials
[17,18].
Photodynamic Therapy (PDT) uses a non-toxic dye molecule or
photosensitizer (PS) that when activated by absorbed photon of
light produces cytotoxic reactive oxygen species (ROS) [19].
Direct tumor killing by ROS, tumor-associated vascular damage
and most notably activation of inflammatory responses make PDT
an effective anti-cancer procedure [20,21]. To date PDT has been
approved by US Food and Drug Administration for use in
bronchial and esophageal cancer and other premalignant and
ophthalmological conditions [22]. Moreover, several other cancers
are under active investigation [23].
PDT is thought to be particularly effective at stimulating an
immune response against a locally treated tumor [20] for the
Introduction
To destroy tumors the immune system uses cytotoxic Tlymphocytes (CTLs) that recognize tumor antigens presented by
major histocompatibility complex (MHC) class I molecules on
the surface of tumor cells [1]. The molecular identity of a number
of these antigens has been recently defined both in mouse and
human tumors [2]. The tumor antigens identified to date have
been broadly divided into following major groups [3]: (i) antigens
encoded by cancer-testis genes expressed in various tumors, but
not in normal tissues, such as the mouse gene P1A and human
genes of the MAGE, BAGE and GAGE families [4,5,6,7,8,9]; (ii)
differentiation antigens of the melanocytic lineage, which are
present on most melanomas but also on normal melanocytes
[9,10,11]; and (iii) antigens that result from tumor-specific
mutations in genes which are expressed in all tissues or come
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PDT Induces Systemic Immunity against Tumor Antigen
The successful use of PDT to induce an effective local
inflammatory response has been demonstrated in several tumor
models [20,39]; however, there is a limited amount of data
recognizing the systemic immunological effects of this local
treatment. In particular, the dependence and involvement of
PDT mediated immunity on expression of tumor antigens has not
been thoroughly established. We used a pair of equally lethal
BALB/c colon adenocarcinomas, CT26 wild-type (CT26WT) and
CT26.CL25 that expressed a tumor antigen, b-galactosidase (bgal) to show that PDT treatment can elicit a systemic antigen/
epitope specific anti-tumor immune response sufficiently robust to
lead to regression of distant, well-established, antigen positive
tumors outside the treatment field.
following reasons. PDT has been shown to effectively engage both
innate and adaptive immune systems in the host’s responses to
cancer [24,25,26]. PDT alters the tumor microenvironment by
stimulating the release or expression of various pro-inflammatory
and acute phase response mediators from the PDT-treated site
[27,28,29,30]. The body recognizes the presence of local trauma
threatening the integrity of the affected site, and releases
proinflammatory mediators to maintain homeostasis [31]. PDT
thereby prompts a powerful acute inflammatory response, causing
accumulation of neutrophils and other inflammatory cells in large
numbers at the treated site and attack tumor cells [28,32]. The
activation of complement system has in particular emerged as a
powerful mediator of PDT anti-tumor effects [33,34,35,36,37].
Complement not only acts as a direct mediator of inflammation
but also stimulates cells to release secondary inflammatory
mediators, including cytokines IL-1b, TNF-a, IL-6, IL-10, GCSF, thromboxane, prostaglandins, leukotrienes, histamine, and
coagulation factors [30].
In addition to stimulating local inflammation, PDT acts
systemically to induce a potent acute phase response. PDT may
also mature and activate dendritic cells and increase their ability to
home to lymph nodes and efficiently present tumor antigens and
prime lymphocytes [38].
Results
PDT treatment leads to cures of antigen expressing
tumors
The employed pair of previously described tumors, namely the
b-gal antigen positive CT26.CL25 and antigen negative counterpart CT26WT cells were characterized by similar in vitro
susceptibility to PDT (Figure 1A) and comparable levels of
MHC class I molecules (Figure 1B). The CT26.CL25 cells
Figure 1. In vitro studies. A. In vitro PDT effectiveness against CT26WT and CT26.CL25 cells. The bars represent standard deviation. B. Histogram
analysis of the levels of MHC I molecules in CT26.CL25 and CT26WT cell lines. (Blue) CT26.CL25 unstained control, (Dark Green) CT26.CL25 Isotype
control, (Purple) CT26.CL25 anti-MCH I, (Black) CT26WT unstained control, (Bright Green) CT26WT Isotype control, (Red) CT26WT anti MHC I. C.
Expression levels of b-gal antigen in CT26WT. D. Expression levels of b-gal antigen in CT26.CL25. E. Scheme of in vivo PDT.
doi:10.1371/journal.pone.0015194.g001
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PDT Induces Systemic Immunity against Tumor Antigen
displayed uniform expression of b-gal antigen (Figure 1C), while
the CT26WT were b-gal antigen negative (Figure 1D).
The scheme of the subsequent set of in vivo experiments is
depicted in Figure 1E. We employed a vascular PDT regimen that
was highly effective in mediating local regression in treated
tumors. PDT produced a local response in all b-gal antigen
negative CT26WT tumors as manifested by a marked reduction in
size lasting until day 18 (Figure 2A). However, local tumor
regrowth occurred relatively quickly and the net result was a
growth delay of only 8–10 days. In marked contrast were the
tumor volumes of b-gal antigen positive CT26.CL25 tumors
treated with PDT (Figure 2B). The reduction in size was complete
beyond day 20 and most importantly 100% of these PDT treated
antigen positive tumors stayed in remission for the whole 90-day
course of observation. Consequently, mice were declared cured
according to the protocol.
To exclude the possibility that the observed difference in antitumor PDT effectiveness between CT26WT and CT25.CL25
could be attributed to the effects of the vector used to induce
expression of b-gal, we created another control cell line CT26neo
and Figure S1 shows the results of PDT treatment of this specially
designed, additional, vector alone control.
PDT cured mice reject rechallenge in an antigen specific
manner
To assess memory immunity we performed rechallenge experiments. Mice bearing antigen positive CT26.CL25 tumors that had
received PDT treatment and remained tumor free for 90 days were
subsequently inoculated with the same antigen positive CT26.CL25
cells into the contralateral thigh. To assess the antigen specificity of
the memory immunity some of the mice that were cured from
CT26.CL25 cells were inoculated with antigen negative CT26WT
cells. More than 95% of mice rechallenged with CT26.CL25 tumors
rejected the tumor challenge and stayed tumor free for another 60
days of observation (Figure 2C), while all antigen negative CT26WT
tumors progressed. Control survival curves of naı̈ve mice bearing
CT26.CL25 or CT26WT tumors demonstrated that the cells used
for rechallenge retained their full tumorigenic potential.
PDT treatment leads to increase in local production of
TNFa and IFNc cytokines in antigen positive tumors
We assessed the extent of local activation of the immune system
by measuring secreted cytokines in the tumor. We observed that
PDT treatment of antigen positive CT26.CL25 (but not antigennegative CT26WT) tumors led to striking and significant
Figure 2. In vivo PDT of tumors (1 leg model). A. Plots of mean tumor volumes in mice bearing CT26WT tumors and B. CT26.CL25 tumors. Points
are means of from 10–15 tumors and bars are SD. C. Kaplan-Meier survival curves of the % of mice cured from CT26.CL25 tumors and rechallenged
either with CT26.CL25 cells or CT26WT cells. Naı̈ve mice are included as a control for tumorigenic properties of the cells. Survival curve for rechallenge
with CT26.CL25 cells is significantly different from the other two curves (P,0.0001). D. Mean levels of cytokines (TNF-alpha, IFN-gamma, IL-2 and IL-4)
measured in the CT26WT and CT26.CL25 tumors 5 days after PDT as well as in control, non treated tumors. *** p,0.001. The bars represent standard
deviation.
doi:10.1371/journal.pone.0015194.g002
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PDT Induces Systemic Immunity against Tumor Antigen
incubated with DimerX loaded with nonapeptide derived from
b-gal antigen (TPHPARIGL peptide). There was a significant
difference (Figure 3D) between binding of b-gal loaded DimerX by
CD8 positive T cells isolated from mice cured from CT26.CL25
tumors and from naı̈ve CT26.CL25 tumor bearing mice. These
results show that PDT does indeed induce recognition of MHC
class I bound epitope derived from b-gal antigen and provide an
explanation for the specific cell lysis found in the chromium release
experiments.
(p,0.001) increases in tumor necrosis factor alpha (TNFa) and
interferon gamma (IFNc) levels (Figure 2D) suggesting the active
involvement of the Th1 arm of adaptive immune response. The
production of IL-2 and IL-4 was not significantly different from
the non-treated control levels.
PDT induced cytotoxic T cells specifically destroy
antigen-positive cancer cells
To confirm that PDT leads to development of b-gal antigen
specific cytotoxic T cells able to specifically lyse tumor cells in an
antigen specific manner we used 51Cr release assay. We harvested
the regional, tumor draining lymph nodes from CT26.CL25 cured
mice five days after tumor rechallenge as well as from control,
tumor-bearing mice 9 days after tumor inoculation. Figure 3A
shows that CTLs from mice cured from antigen positive
CT26.CL25 tumors with PDT displayed significantly more
specific lysis at effector to target ratios of 25:1 and 50:1 against
CT26.CL25 targets than they did against antigen negative
CT26WT targets (P,0.05) or irrelevant, antigen negative
EMT6 targets (P,0.001). Likewise, lymphocytes from
CT26.CL25 tumor bearing mice also showed significantly less
specific lysis against CT26.CL25 targets than did CTLs from
CT26.CL25 PDT cured mice (P,0.05).
PDT of antigen-positive tumors leads to destruction of
distant, untreated, established, antigen-positive tumors
To further evaluate whether PDT treatment can elicit b-gal
antigen specific systemic immune response strong enough to
destroy distant, established, non-treated tumors we performed
PDT in mice bearing two bilateral tumors. In this model only one
tumor was illuminated, while the contralateral tumor was shielded
from light. The antigen negative CT26WT tumors that received
treatment could not be followed for the long-term outcome
because all untreated, contralateral tumors continued their growth
uninterrupted, confirming the lack of any PDT induced antitumor immunity (Figure 4B). The untreated control bilateral
CT26WT tumors grew equally well leading to mouse sacrifice
when tumors reached 1 cm in diameter (Figure 4A). Mice bearing
b-gal antigen positive CT26.CL25 tumors in both legs, which had
only one tumor illuminated, demonstrated a remarkable and
interesting response: the PDT treated tumors regressed in all cases;
in 9 out of 10 mice the distant untreated tumors also shrank and
disappeared for at least 20 days, while in one mouse the tumor
continued growth unabated. In 6 out of 9 mice the tumor
regression of their contralateral tumors lasted beyond day 20 and
PDT elicits development of epitope specific CD8+ T cells
In order to demonstrate that PDT of antigen positive
CT26.CL25 tumors can lead to recognition of specific epitopes
derived from particular tumor antigen we used Dimer X staining
[40]. The lymph node cells isolated from mice either cured from
antigen positive CT26.CL25 mice (Figure 3B) or control, nontreated CT26.CL25 tumor bearing mice (Figure 3C) were
Figure 3. Analysis of antigen and epitope specificity of observed PDT induced immune response. A. Percentage of specific lysis of target
cells (CT26.CL25, CT26WT or EMT6 as an irrelevant target control) by CTLs isolated from either CT26.CL25 PDT cured or control CT26.CL25 tumor
bearing mice (3–4 mice per group). Data are representative of 3 independent experiments. * P,0.05 compared to CT26.CL25 cured CTLs against
CT26WT targets, and compared to CTLs from CT26.CL25 tumor bearing mice. ## P,0.001 compared to CT26.CL25 cured CTLs against EMT6 targets.
The bars represent standard deviation. B. Lymph node cells isolated from PDT treated mice curedfrom antigen positive CT26.CL25 tumors 5 days after
rechallenge incubated with DimerX loaded with TPHPARIGL peptide derived from b-gal antigen or empty DimerX, and either FITC-detection antibody
or FITC isotype control. Additionally cells were stained for CD8 expression to assess percentage of CD8-DimerX-FITC double positive cells. C. Lymph
node cells from CT26.CL25 control tumor bearing mice incubated with DimerX loaded with TPHPARIGL peptide derived from b-gal antigen and FITCdetection antibody. Additionally cells were stained for CD8 expression to assess percentage of CD8-DimerX-FITC double positive cells. D.
Quantification and statistical analysis of the FACS plots described above (6 mice per group). The bars represent standard deviation.
doi:10.1371/journal.pone.0015194.g003
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PDT Induces Systemic Immunity against Tumor Antigen
Figure 4. In vivo PDT of tumors (2 leg model). Time courses of individual tumor volumes in mice with two similar bilateral or mismatched
tumors in right and left legs. A. Bilateral CT26WT tumors, right leg treated with PDT (n = 10); B. Bilateral CT26WT tumors, untreated (n = 5); C. Bilateral
CT26.CL25 tumors, right leg treated with PDT (n = 10); D. Bilateral CT26.CL25 tumors, untreated (n = 5). E. Kaplan-Meier survival curves of the % of
mice with tumors less than 1-cm diameter in five groups of mice. Three groups had two similar bilateral CT26.CL25 tumors (one group was untreated,
one group had right leg tumor treated with PDT and one group had right leg tumor surgically removed). Two further groups had two bilateral
CT26WT tumors (one group was untreated, and the other group had the right leg tumor treated with PDT). The survival curve of the mice with
bilateral CT26.CL25 tumors treated with PDT is significantly different from the other survival curves (P,0.0001). F. Mismatched tumors. CT26WT and
CT26.CL25 tumors, CT26WT treated with PDT (n = 5). G. Mismatched tumors. CT26WT and CT26.CL25 tumors, CT26.CL25 treated with PDT (n = 5).
doi:10.1371/journal.pone.0015194.g004
CT26WT tumors treated with PDT and the other group had only
CT26.CL25 tumors treated with PDT. The PDT treated tumors
showed the expected PDT response (more pronounced in the case
of CT26.CL25), but since there were no effects on the size or
growth rate of the contralateral untreated tumors in either case
(Figure 4 F&G) mice could not be followed for long-term outcome.
was permanent. In 2 out of 9 mice the untreated, contralateral
tumors recurred about day 30 and in one mouse the untreated,
contralateral tumor regrew briefly about day 50 before also
regressing permanently. The growth curves of these tumors are
shown in Figure 4D and the corresponding growth curves of
untreated bilateral CT26.CL25 tumors are shown for control
purposes in Figure 4C. Kaplan-Meier curves for the percentage of
mice with both tumors smaller than 1-cm in diameter are shown in
Figure 4E.
It was considered possible that the simple mechanical removal
of one of the tumors by the ablative function of PDT could affect
the growth of the contralateral one. To test and exclude this
possibility a group of mice bearing bilateral antigen positive
CT26.CL25 tumors had the right-leg tumor surgically removed at
the same time as PDT was carried out to other groups, but this
treatment had no effect on the progression of the contralateral
tumors (Figure 4E). The survival curve for the CT26.CL25 PDT
treated group was significantly different from all other experimental groups (P,0.0001, log-rank test).
To further investigate whether observed destruction of contralateral, established, non-treated tumors was antigen specific we
carried out experiments with two groups of mice that each had two
mismatched tumors, antigen negative CT26WT in left leg and
antigen positive CT26.CL25 in right leg. One group had only
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Activated cytotoxic T cells infiltrate antigen-positive PDT
treated and non-treated, contralateral tumors
To further confirm the involvement of the immune system in
the observed PDT response and the destruction of contralateral,
established, non-treated tumors we performed immunohistochemical staining for LAMP-1 (CD107a) presence as a marker for
intratumoral activated cytotoxic T cell infiltration [41,42]. We
observed that CT26.CL25 non-treated, antigen positive control
tumors demonstrated some staining (Figure 5A), while PDT
treated CT26.CL25 tumors examined 5 and 16 days after PDT
treatment revealed pronounced T cell infiltration (Figure 5C&E).
In addition, the LAPM-1 staining demonstrated that contralateral
(Figure 5D&F) antigen positive CT26.CL25 tumors are also
heavily infiltrated by LAMP-1 positive T cells. Moreover, there
was noticeable increase in T cell infiltration/LAMP-1 staining
between day 5 and 16 which corresponded well with the observed
decrease in tumor size.
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PDT Induces Systemic Immunity against Tumor Antigen
Figure 5. Immunohistochemical staining for LAMP-1 (CD107a) of CT26.CL25 tumors. A. non-treated control. B. negative control for
staining. C. CT26.CL25 PDT treated tumors day 5. D. CT26.CL25 non-treated, contralateral tumors day 5. E. CT26.CL25 PDT treated tumors day 16. F.
CT26.CL25 non-treated, contralateral tumors day 16. Analysis of b-gal antigen expression and loss by X-gal staining. G. CT26WT control tumors
negative for b-gal antigen, H. CT26.CL25 non-treated control tumors which show robust blue positive staining for b-gal antigen. I. CT26.CL25 nontreated, contralateral tumors that escaped immune surveillance and continued to grow. They show significantly decreased staining for b-gal antigen.
doi:10.1371/journal.pone.0015194.g005
PDT treatment provided good local response, but it did not affect
the growth of the non-treated, contralateral, b-gal antigen positive
CT26.CL25 tumors. In figure 6C we compared survival of
immunocompetent BALB/c and immunocompromised BALB/c
Nu/Nu mice bearing CT25.CL25 as well as CT26WT tumors. As
can be seen PDT treatment of CT25.CL25 tumors in immunocompetent mice resulted in 100% survival. However, the PDT
treatment of CT26.CL25 tumors growing in immunocompromised mice failed to produce any cures and the recurrence of
CT25.CL25 tumors in BALB/c Nu/Nu mice closely resembled
the recurrence of CT26WT tumors in BALB/c mice. These
results provide strong evidence that the curative effects observed in
case of antigen positive CT26.CL25 tumors were mediated by
PDT activated, antigen specific immune response, and that the
lack of functional adaptive immune system abrogates this effect.
Tumors escape PDT mediated immune surveillance by
decreasing antigen expression
It was possible that the reason why 3 out of 10 contralateral
tumors escaped from PDT mediated immune recognition and
elimination could be due to the loss of the expression of the b-gal
antigen under the pressure of immune destruction. We therefore
harvested the contralateral tumors that progressed after PDT and
stained them for b-gal antigen presence. We observed that indeed
tumors which escaped immune destruction had significantly lower
levels of b-gal antigen (compare Figures 5H&I).
Lack of adaptive immune system abrogates PDT
anti-tumor effects
To confirm that the observed PDT effects are indeed due to the
activation of the immune system we repeated the experiments with
b-gal antigen positive CT26.CL25 tumors in immunocompromised BALB/c Nu/Nu mice. In a one-leg model PDT produced a
local response similar to that observed in antigen negative
CT26WT tumors, but no permanent cures were observed
(Figure 6A). To provide additional evidence for the involvement
of the immune system in the destruction of the non-treated,
contralateral, b-gal antigen positive CT26.CL25 tumors, we also
repeated the PDT experiments in immunocompromised mice
bearing bilateral CT26.CL25 tumors. As can be seen in Figure 6B,
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Discussion
In this study we have employed a pair of previously described
tumors, CT26.CL25 transduced with lacZ gene to stably express a
model tumor antigen (b-gal) and its wild type counterpart CT26.
This pair of tumors allowed us to design a study model closely
resembling the clinical situation to investigate the importance of
the antigen presence and differences of PDT-induced immune
reaction between antigen expressing and antigen-negative cancer
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PDT Induces Systemic Immunity against Tumor Antigen
Figure 6. Lack of adaptive immune response abrogates PDT anti-tumor effects. A. Tumor volumes of CT26.CL25 tumors subjected or not
to PDT in BALB/c Nu/Nu immunocompromised mice. The bars represent standard deviation. B. Tumor volumes of bilateral CT26.CL25 tumors
subjected or not to PDT in BALB/c Nu/Nu immunocompromised mice. The bars represent standard deviation. C. Kaplan-Meier analysis comparing the
% of surviving BALB/c and BALB/c Nu/Nu mice bearing CT26.CL25 of CT26WT tumors subjected to PDT. Non-treated BALB/c Nu/Nu mice bearing
CT26.CL25 are included for control (n = 5).
doi:10.1371/journal.pone.0015194.g006
response was only observed in the antigen positive tumors
emphasizes the importance of the presence of a tumor antigen
capable of being efficiently recognized by cytotoxic effector T cells.
These data demonstrate that PDT induced CTLs discriminate and
target antigen expressing tumor as well as that PDT immunity
depends on the presence of tumor antigen.
The expression of b-gal antigen in mouse tumors has been
previously described and it was found that in certain circumstances
it could act as a potent immunostimulant leading to generation of
cytotoxic T lymphocytes [50,51]. The reported antitumor effects
of vaccination protocols with vectors carrying the b-gal gene had
to be significantly enhanced by the co-administration of certain
cytokines [50] or by the viral vectors simultaneously encoding
cytokine genes [52]. The active immunity against established
tumor produced by vaccinationin the absence of additional
interventions, failed to have an impact on tumor burden.
Therapeutic responses in tumor bearing animals could only be
improved however, when particular cytokines (rhIL-2, rmIL-6,
rhIL-7, and rmIL-12) were given following vaccine administration.
However, it has never before been reported that a single
immunotherapy treatment of the b-gal positive tumor can lead
to total tumor rejection [53]. Our data strongly suggest that
applying a single PDT treatment to an antigen-expressing tumor
may not only destroy the primary tumor, but also induce a
systemic immune response capable of destroying distant antigen
positive metastases.
One interesting observation from our study that needs
explanation is the failure of PDT to induce anti-tumor immunity
in CT26WT tumors. PDT of CT26 tumors growing in
immunocompetent mice followed by intratumoral injection of
immature dendritic cells has been previously shown to produce
some immune response leading to a slower growth of a tumor
rechallenge, and this combination therapy was able to generate
CTLs capable of lysing tumor cells ex vivo [21,38,54]. There are
also several reports that CT26 tumors in its wild-type state do
express tumor antigens [55] in particular a single peptide known as
AH-1, a non-mutated nonamer derived from the envelope protein
(gp70) of an endogenous ecotropic murine leukemia provirus [55].
Nevertheless, most authors agree that in practice CT26WT
tumors in BALB/c mice generally evade the immune response due
to the presence of CD4+CD25+ T-regulatory suppressor cells
cell lines, otherwise being identical. In this model both wild type
and b-gal tumors were equally lethal, suggesting that the level of bgal expression was low enough to allow tumor to grow without
triggering any clinically significant immune response, a situation
often observed in cancer patients [43]. It was only when PDT was
applied that the significant differences in the therapeutic outcome
and the elicited immune response were observed.
The present study shows that PDT can induce a highly potent
antigen specific immune response capable of inducing memory
immunity that enables mice to reject a tumor rechallenge with the
same antigen positive tumor from which they were cured. The in
vivo PDT-induced immune response led to an increased release of
TNFa and IFNc cytokines within the treated tumors. The CTLs
from PDT treated mice bearing antigen positive CT26.CL25
tumors were capable of causing specific lysis of antigen positive
target cells and bind the immunodominant peptide epitope
derived from b-gal antigen restricted by MHC class I haplotype
H2Ld. PDT induced immune response was also capable of causing
regression of distant established tumors that received no treatment.
The robust infiltration of PDT treated and non-treated, contralateral tumors by activated, antigen specific effector CTLs has also
been confirmed. However, the regression of distant tumors
occurred in only 70% of mice and the reason why some tumors
escaped from immune recognition and elimination was shown to
be the loss of expression of the tumor antigen. This is the first time
that the escape from PDT induced immune surveillance due to the
loss of tumor antigen has been demonstrated. However, this
phenomenon has been previously described [44,45,46,47] also in
the case of CT26.CL25 model [48] when lung metastases from
CT26.CL25 tumors, which had escaped from immune control
after virus-mediated vaccination, were shown to have reduced bgal activity. Most importantly we showed that the observed PDT
anti-tumor effects are completely abolished when there is no
functional adaptive immune response.
Our findings are in accordance with recently published study by
Kabingu et al. [49] where it was shown that PDT could lead to
immune recognition of hedgehog-interacting protein 1 (Hip1)
antigen in patients with basal cell carcinoma. These observations
show that PDT has the capability to be a local cancer therapy that
could be usefully applied even when the primary tumor has spread
at the time of treatment. The fact that this potent immune
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PDT Induces Systemic Immunity against Tumor Antigen
CT26neo cell line was provided by Dr. Andrew Kung from
Department of Pediatric Oncology, Dana-Farber Cancer Institute,
Boston, MA. VSVG-pseudotyped retrovirus was packaged by
triple transfection of pLNCX-neo, pMD-MLV, and pMD-G
(Richard Mulligan, HHMI, Boston, MA) into 293T cells.
CT26WT cells were infected with filtered retroviral stocks at a
multiplicity of infection of 10 in the presence of 8 mg/ml of
polybrene. CT26neo and CT26.CL25 cells were cultured in
constant presence of 500 mg/mL G418 antibiotic (Sigma, St Louis,
MO) in order to maintain constant expression of the vector.
[56], down-regulation of gp70 production [57], or the presence of
immune tolerance [58]. Recently, a paper by McWilliams et al.
[59] described the expression of gp70 mRNA in several tissues of
BALB/c mice resulting in immunologic tolerance that affects
antitumor immunity. In view of these reports it is possible that
gp70 antigen in CT26WT tumors behaves like self antigen and
therefore an intervention targeting T regulatory cells may
potentiate PDT immune response in this model. We have
previously shown that low dose cyclophosphamide (CY) can
deplete T regulatory cells and augment PDT immunity in J774
tumors [60] and we subsequently found out that the combination
of the described low-dose regimen of CY and PDT may lead to
significant numbers of cures in the CT26WT model (unpublished
data).
The employed tumor antigen model somewhat differs from
many naturally occurring cancer antigens, including the fact that
the expression of the antigen is limited to tumor tissue or that the
immune response is studied in established transplanted tumors and
not in metastatic setting. Many would argue that the true test of a
systemic immune response is the effects on distant metastases.
However, there are reports [61,62] where the effectiveness of
immunotherapy was demonstrated both in models of metastatic
disease and in animals bearing established tumors. Furthermore,
the destruction of an untreated established tumor may appear as a
more severe test of active immune response than metastatic disease
which, microscopic in nature, may be more easily penetrated by
tumor specific immune cells. Notwithstanding with the restrictions
of the selected model the presented results however, are a
straightforward demonstration of the importance of tumor
antigens in promoting immune rejection of tumors and in this
regard they may have significant implications for the design of
clinical protocols using PDT to treat human cancers. We believe
that more investigators should consider whether antigen-specific
immune response is involved in patients receiving PDT for cancer.
Consequently, the results presented in this study have led us to
explore the effects of PDT employing tumors expressing clinically
relevant tumor antigens like P815 mastocytoma expressing murine
homologue of cancer testis antigen P1A [63] or pancreatic
adenocarcinoma Panc02 expressing human carcinoembryonic
antigen [64]. The preliminary results obtained in these models
are highly encouraging and in agreement with the presented data
(unpublished results).
In conclusion, we have shown that an effective vascular PDT
regimen that can reliably produce local tumor destruction can also
induce potent, systemic, antigen specific anti-tumor immunity.
The observed immunity was capable of causing regressions and
cures in distant, established, antigen positive tumors outside the
illumination field, and also of inducing long-term immune
memory and resistance to rechallenge. This tumor-destructive
effect was mediated by tumor antigen specific cytotoxic T-cells
that recognize the immunodominant epitope of b-gal antigen and
are induced after PDT. These data encourage clinical trials of
PDT in patients with tumor types known to express tumor
associated antigen (melanoma, renal cell carcinoma etc).
Cell X-gal staining
To detect b-gal antigen CT26.CL25, CT26WT and CT26neo
cells were fixed with X-gal Fix buffer (0.1 M phosphate buffer
(pH 7.3) supplemented with 5 mM EGTA (Sigma), pH 7.3,
2 mM MgCl2 and 0.2%glutareldahyde (Sigma)) for 15 min, than
washed twice (5 minutes each) with X-gal Wash buffer (0.1 M
phosphate buffer (pH 7.3) supplemented with 2 mM MgCl2). Next
the X-gal staining buffer containing 1 mg/mL X-gal (0.1 M
phosphate buffer (pH 7.3) supplemented with 2 mM MgCl2,
5 mM potassium ferrocyanide and 5 mM potassium ferricyanide
was added and cells were incubated overnight.
Tumor X-gal staining
We used a modified method of staining mouse tumors with Xgal [65]. Briefly to detect beta-gal expression non-treated
CT26WT, CT26.CL25 and contralateral CT26.CL25 tumor
samples that escaped PDT induced immunity were harvested
when tumors reached 1.5 cm in diameter, fixed with Fix buffer
(0.1 M phosphate buffer (pH 7.3) supplemented with 5 mM
EGTA (Sigma), pH 7.3, 2 mM MgCl2 and 0.2% glutaraldahyde
(Sigma)) for 15 min and washed twice (5 minutes each) with X-gal
Wash buffer (0.1 M phosphate buffer (pH 7.3) supplemented with
2 mM MgCl2). Next the X-gal staining buffer containing 1 mg/ml
of X-gal (0.1 M phosphate buffer (pH 7.3) supplemented with
2 mM MgCl2, 5 mM potassium ferrocyanide (K4Fe(CN)6-3H2O)
and 5 mM potassium ferricyanide (K3Fe(CN)6) was added and
tumors were incubated overnight at 37uC. The pictures were taken
with the Olympus SLR digital camera.
Flow cytometry analysis of MHC class I molecules levels
CT26.CL25 and CT26WT cells were fixed and incubated at
room temperature for 1 h with PE-Conjugated anti H2-Dd
antibody (BD Pharmingen). PE Isotype antibody and unstained
cells were used as controls. Next cells were washed twice in 1 ml of
PBS and analyzed on FACScalibur (BD).
Photosensitizer and light source
Materials and Methods
Liposomal benzoporphyrin derivative mono acid ring A
(Verteporfin for injection, BPD, QLT Inc, Vancouver, BC,
Canada) and was prepared by diluting the powder to a
concentration of 0.3 mg/mL in sterile 5% dextrose. A 1W 690nm diode laser (B&W Tek Inc., Newark, DE) was coupled into a
0.8-mm diameter fiber and a lens was used to obtain a uniform
spot.
Cell lines
In vitro PDT
CT26 wild type (CT26WT) and CT26.CL25 cell lines (ATCC,
Mannassas, VA). were cultured in RPMI medium with Lglutamine and NaHCO3 supplemented with 10% heat inactivated
fetal bovine serum, penicillin (100 U/mL) and streptomycin
(100 mg/mL) (all from Sigma, St Louis, MO) at 37uC in 5%
CO2 in 75 cm2 flasks (Falcon, Invitrogen, Carlsbad, CA).
10000 CT26WT, CT26.CL25 and CT26neo cells were plated
per well in 96 well plates and incubated for 1-h with 200 nM BPD.
%. After incubation the medium was replaced with 200 mL of
fresh medium and PDT was performed. 690 nm laser light dose
was varied and fluences of 0 (dark toxicity) to 2 J/cm2 were
delivered at an irradiance of 50 mW/cm2 to each well separately
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PDT Induces Systemic Immunity against Tumor Antigen
sacrificed 9 days after tumor injection were homogenized with a
pellet pestle (Kontes Glass Co, Vineland, NJ) and passed through a
70 mm mesh nylon cell strainer (BD Falcon) to make single cell
suspensions.
(4 wells represented a group). Controls entailed cells with no
treatment and cells with light alone at the highest fluence or with
photosensitizer alone. At the completion of the illumination, the
plates were returned to the incubator for 24 h before initiating
further studies. A 4-h MTT colorimetric assay [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was used
that measures mitochondrial reductase activity. This assay
correlates well with colony-forming assays as a measure of cell
viability, as has been described previously. The absorbance for
MTT assay was read at 560 nm.
Chromium release assay
Cytotoxicity was measured by 51Cr release assay. Lymphocyte
suspensions (0.1 mL) were dispensed to wells of U-bottom 96-well
microtiter plates (six wells replicates for each effector:target (E:T)
ratio). One million target cells (CT26WT, CT26.CL25 or EMT6)
were labeled for 2 h with 100-mCi of 51Cr (NEN Perkin Elmer,
Waltham, MA), washed and then 10,000 target cells were mixed
with effector CTLs at various E:T ratios and incubated for 4-h at
37uC, 5% CO2. 40 mL of supernatant were mixed with 150 mL of
scintillation cocktail (Optiphase Supermix. NEN Perkin Elmer)
and a beta scintillation was read with plate reader (MicroBeta
Trilux Model 1450, Wallac-Perkin-Elmer). The final percentage of
specific lysis was calculated as follows: {test 51Cr released –
spontaneous 51Cr released}/{maximum 51Cr released – spontaneous 51Cr released}. The maximal release was obtained by
incubation of target cells in 0.5% SDS.
Animal tumor model
BALB/c and BALB/c Nu/Nu mice (6–8 weeks old) were
purchased from Charles River Laboratories (Boston MA). All
experiments were carried out according to a protocol approved by
the Subcommittee on Research Animal Care (IACUC) at MGH
and were in accord with NIH guidelines. Mice were inoculated
with 350,000 cells subcutaneously into the depilated right thigh.
Two orthogonal dimensions (a and b) of the tumor were measured
2–3 times a week with vernier calipers. Tumor volumes were
calculated as follows, volume = 4p/36 [(a+b)/4]3. When tumors
reached a diameter of 5–7 mm (9 days after inoculation) PDT was
performed.
Dimer X staining
The H2Ld specific b-gal peptide TPHPARIGL [51] was
synthesized by the MGH Institutional Peptide Synthesis Core.
4 mg of soluble dimeric mouse H-2Ld:Ig fusion protein (BD
Biosciences, San Jose, CA) was loaded with a 640-fold molar
excess of TPHPARIGL peptide overnight at 37uC. Lymphocytes
from CT26.CL25 immune or tumor-bearing mice were incubated
with staining cocktail (H-2Ld:Ig peptide loaded, PerCP labeled
anti-CD8a clone 53-6.7 (BD Pharmingen) and FITC-labeled rat
anti-mouse IgG1 clone A85-1 (BD Biosciences) for 1 h at room
temp, washed and FACS analysis was performed. (FACScalibur,
BD Biosciences). The appropriate FITC and PerCP secondary
antibody isotype controls as well as staining with empty H-2Ld:Ig
fusion protein was performed. FACS scattergrams were analyzed
by first gating for size and CD8 expression on FL3 vs FSC dot plot
and next by re-plotting CD8 positive cells on FL1 vs FL3 dot plot
to assess percentage of CD8-DimerX-FITC double positive cells.
PDT and tumor response
Tumor bearing mice were anaesthetized with i.p. injection of
87.5 mg/kg of ketamine and 12.5 mg/kg xylazine and BPD
(1 mg/kg in 5% dextrose solution) was administered i.v. via the
supraocular plexus. Control mice received 5% dextrose only.
Fifteen minutes after BPD injection illumination was performed. A
total fluence of 120 J/cm2 was delivered at a fluence rate of
100 mW/cm2. The mice were sacrificed when any of the tumor
diameters exceeded 1.5 cm (1 cm diameter for 2 legs. and
rechallenge models).
Rechallenge
Mice surviving ninety days after PDT were subsequently
rechallenged with 350,000 cells of CT26.CL25 or CT26WT in
the contralateral thigh and monitored for another 60 days. Naı̈ve
control mice were inoculated with the same sample of cells to
confirm tumorigenicity.
PDT in bilateral tumor model
Mice were inoculated in both legs either with CT26WT,
CT26.CL25 or one CT26WT and one CT26.CL25 tumors and
two equal sized tumors reliably grew. After BPD injection only one
tumor received light treatment. Ten mice with bilateral
CT26.CL25 tumors had their right legs amputated above the
tumor at the same day as PDT was performed. This procedure
was done under anesthesia, using a scalpel, the femoral artery was
isolated, sutured and cut with VICRIL 7-0 synthetic absorbable
suture (ETHICON INC. NJ), the bone was cut after hemostasis.
The wound was closed in two planes, first muscle using VICRIL 70 suture, the second plane was skin closed using black
monofilament Nylon nonabsorbable surgical suture (ETHICON,
INC). All these procedures were done following aseptic techniques.
Cytokine production in excised tumors
The levels of cytokines (TNF-alpha, IFN-gamma, IL-2 and IL4) were measured in the CT26WT and CT26.CL25 tumors
harvested 5 days after PDT as well as in control, non-treated
tumors. We used a mouse cytokine capture bead assay (BDTM
Mouse Th1/Th2 Cytokine Kit, Becton-Dickinson, San Diego,
CA). The assays were performed according to the manufacturer’s
instructions. In brief, tumor samples were homogenized in glass
homogenizer and centrifuged to collect supernatants. A 50 mL
aliquot of the supernatant of each sample was stained with a
suspension of mouse cytokine beads and the phycoerythrin (PE)
detection reagent. After 2 hr of incubation, samples were washed
and then analyzed by flow cytometry and CBA software (BD
Biosciences). Mouse Th1/Th2 standards provided with the kit
were appropriately diluted and used in parallel to samples for
preparation of the standard curves. Two different samples were
used for different tumors and each group was repeated twice.
Immunohistochemistry
Formalin-fixed, paraffin-embedded sections of non-treated
control, PDT treated and non-treated contralateral CT26.CL25
tumors harvested on day 5 and 16 of the experiment were
sectioned serially (5 mm). Slides were deparaffinized, subjected to
heat-based antigen retrieval (BD Pharmingen) and stained with
Vectorstain ABC kit (Vector Laboratories, Inc, Burlingame, CA).
First slides were incubated with 10% normal rabbit serum for 20
Lymphocyte preparation
Inguinal lymph nodes from tumor-immune mice sacrificed five
days after CT26.CL25 rechallenge and from tumor-bearing mice
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PDT Induces Systemic Immunity against Tumor Antigen
minutes at room temperature (RT) and next a 1:200 dilution of rat
monoclonal anti LAMP-1 antibody in 1% BSA in PBS (Santa
Cruz; Santa Cruz, CA) was added for overnight incubation at 4uC.
For negative staining control 1% BSA in PBS was used instead of
primary antibody. On the following day a biotinylated secondary
rabbit anti-rat antibody was added for 30 minutes at RT and
following wash with PBS ABC reagent was added for 45 minutes
at RT. Staining was visualized using Vector NovaRed, and
hematoxylin was used as a nuclear counterstain.
PDT tumor volumes of individual mice in the PDT group are
presented. The one mouse that was cured from CT26neo failed to
reject a rechallenge with CT26neo (data not shown).
(DOC)
Acknowledgments
We are grateful to QLT Inc (Vancouver, Canada) for the generous gift of
BPD. We are grateful to Dr. Andrew Kung from Department of Pediatric
Oncology, Dana-Farber Cancer Institute, Boston, MA for providing
CT26neo cell line.
Statistics
All values are expressed as 6 standard deviation and all
experiments were repeated at least twice with comparable results.
Differences between means were tested for significance by one-way
ANOVA. Survival analysis was performed using the KaplanMeier method. P-values of ,0.05 were considered significant.
Author Contributions
Conceived and designed the experiments: PM AS MXW MRH.
Performed the experiments: PM AS. Analyzed the data: PM MRH.
Contributed reagents/materials/analysis tools: MXW. Wrote the paper:
PM MRH.
Supporting Information
Figure S1 Tumor volumes of CT26neo tumors subjected
or not to PDT. Due to variable response of CT26neo tumors to
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